PROJECT SUMMARY Pathology stemming from excess ectopic bone formation, or trauma-induced heterotopic ossification (HO), presents a substantial barrier to recovery in 20% of patients with hip replacements, musculoskeletal trauma, spinal cord injury, amputations and burn injuries. Patients with HO experience chronic pain, restricted joint function, and open wounds; they often undergo surgical procedures to excise the offending bone, but these procedures fail to reverse the joint contractures and restricted range of motion and can lead to recurrence. With these limitations in our understanding of HO and our inability to prevent its development, we set out to clarify the cells responsible (mesenchymal cells and macrophages) and primary signaling pathway involved. Whole transcriptome sequencing and immunofluorescent imaging of the early HO site have demonstrated a marked increase in Transforming Growth Factor-beta activating kinase 1 (TAK1) signaling in the mesenchymal cells. However, the stage of mesenchymal cell differentiation in which TAK1 is necessary and what cells stimulate mesenchymal cell TAK1 remains unknown. Preliminary data demonstrate that gene and therapeutic knockdown of TAK1 mitigates early mesenchymal condensation and differentiation responsible for HO. Importantly, we have also demonstrated the central role of macrophages and their activation of TAK1 in mesenchymal cells. We plan to validate a novel cell specific drug delivery system to block secretion of a primary ligand (Transforming Growth Factor 1?) from a primary cell (macrophage) responsible for HO. The following aims are designed to test our hypothesis that the overall treatment paradigm of HO will be improved with early detection using high frequency spectral ultrasound and timed, pathway and cell specific inhibition of TAK1 signaling. Aim 1: To define the pathway of TAK1 signaling in mesenchymal cells after trauma and to validate novel TAK1 pathway inhibitors to prevent heterotopic ossification. This aim will demonstrate that TAK1 signaling is upregulated during mesenchymal condensation and that genetic knockout of TAK1 specifically in mesenchymal cells or early TAK1 pharmacologic inhibition with small molecule NG-25 will mitigate HO through an Arkadia/SMAD7 mediated process. This aim will utilize novel imaging to allow detection of early pre-HO changes and validate an early timed treatment strategy to prevent HO. Aim 2: To define the role of macrophage-specific TGF?1 production on HO and to validate novel microparticles that silence Tgfb1 specifically in macrophages. This aim will demonstrate that injury site macrophages and their production of TGF?1 is critical for ectopic mesenchymal cell TAK1 signaling, chondrogenesis and HO. This aim will also optimize microparticles for macrophage-specific uptake and drug delivery to administer Tgfb1 siRNA and prevent HO.